PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5’-splice site (5’SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5’SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Figure S1: Generation and characterisation of RP13-iPSCs.A) Representative microphotographs showing typical iPSC morphology in RP13-iPSCs, scale bars 100 µm; B) Flow cytometric analysis of TRA-1-60 and NANOG.All the iPSC lines show single peaks of positive populations accounting for more than 95% of all the cells examined.Representative images of 3 replicates are shown with the red shaded area indicating the antibody immunostained cells and the blue shaded area the isotype control-stained cells; C) Verification of Sendai virus genome (SEV) and the transgenes (KOS, KLF4, C-MYC) clearance by RT-PCR; D) Genome stability analysis of iPSCs carried out with the hPSC Genetic Analysis kit showing comparable copy number with control in all observed loci except for RP13-1A1, which showed a decreased copy number in the Chr12p region as marked by *.Representative DNA sequence of RP13-iPSC and isogenic CRISPR/Cas9 control showing the correction of heterozygous mutation to the homozygous wild type nucleotide: N indicates where a nucleotide was not automatically assigned, and R indicates the base could be either C or A; D) Representative DNA sequences of all five tested off-target sites showing no deviations from the wild type sequence; E) Karyostat genome stability assay excluded chromosomal aberrations in CRISPR/Cas9 corrected iPSC lines.The whole genome view displays all somatic and sex chromosomes in one frame with high level copy number.A value of 2 represents a normal copy number state (CN = 2), value of 3 represents chromosomal gain (CN = 3), value of 1 represents a chromosomal loss (CN = 1).The pink, green and yellow colours indicate the raw signal for each individual chromosome probe, while the blue represents the normalized probe signal used to identify copy number and aberrations (if any); F, G) Table and accompanying plot of results from PluriTest assay.The red and blue areas of the plot show how the tested samples compare with reference pluripotent and non-pluripotent cell types, respectively.PluriCor and NovCor were determined using an algorithm that integrates gene expression data to authenticate pluripotency status.In brief, PluriCor is a score of pluripotency whereas NovCor increases when non-pluripotent cell types are also present.A non-iPSC sample was used in this experiment to serve as a negative control for non-pluripotency.Bar chart of mitochondria number per cell showing significant (p<0.01)decrease in RP13 (10.4±0.3)compared to RP13-Cas9 iPSC-RPE cells (16.9±0.7);D) Bar chart showing unaffected basal membrane linearity of RP13 (0.95±0.01) and RP13-Cas9 (0.86±0.07) iPSC-RPE.TEM image shows infoldings of basal cell membranes.Cell width was measured as a linear distance (D), membrane by freehand trace (d), where linearity=(d/D); E) The mean surface area of RP13 (101± 7µm 2 ) and RP13-Cas9 (127±32 µm 2 ) iPSC-RPE; F) Bar chart showing unaffected median vacuole counts per cell for RP13 (2.1±0.4) and RP13-Cas9 iPSC-RPE (2.5±1.2).TEM images highlight relevant features in RP13 (red) and RP13-Cas9 (green) in (A, B, D); G) Bar chart showing unaffected density of stage III melanosomes per cell in RP13 (14.2±1.9) and RP13-Cas9 iPSC-RPE (11.3±0.67);H) Bar chart showing unaffected density of stage IV melanosomes per cell in RP13 (0.5±0.5) and RP13-Cas9 iPSC-RPE (0±0); I) Bar chart showing unaffected density of melanolipofuscin-like complexes per cell in RP13 (4.8±0.6) and RP13-Cas9 iPSC-RPE (5.0±1.5);J) Bar chart showing unaffected density of melanolysosome-like complexes per cell in RP13 (20.2±1.7) and RP13-Cas9 iPSC-RPE (23.0±2.5).Representative images are shown on right-hand side of (G, H, I, J); K) Bar charts showing no significant difference in the number and area of collagenous deposits per cell in RP13 and RP13-Cas9 iPSC-RPE.TEM image of collagenous deposits (red outline) with characteristic banded appearance; L) Bar charts showing no significant difference in the number and area of amorphous deposits per cell in RP13 and RP13-Cas9 iPSC-RPE.TEM image of an amorphous deposit (red outline), showing rounded electron dense appearance.Scale bars: 2 µm (A, B, C, D), 0.5 µm (D, E, F, G) and 1 µm (I, J).C-L, n = 3, where n is the median value per donor calculated from 10 cells per group.Results are presented as mean ± SEM.Statistical significance was assessed using a 2-tailed paired t-test.

Figure S5
. Single cell (sc) RNA-Seq of day 210 RP13 and control ROs.A) UMAP visualisation of scRNA-Seq data showing the presence of all retina cell types (late retinal progenitor cells, transient neurogenic progenitors, mitotic cells, rods, cone, amacrine, retinal ganglion, horizontal, bipolar and Muller glia cells), alongside small fractions of RPE cells, astrocytes and fibroblasts; B) Side-by-side comparison of cell type percentage in control and RP13 organoids; C) A higher fraction of degenerating rods is present in RP13 organoids, data shown as mean ± SEM, * p < 0.05, pairwise Student t-test; D, E) Volcano plots showing differentially expressed genes between RP13 cones (D) and rods (E) and isogenic control cells.Grey points represent genes that do not exceed the -log10 p-value or log2 fold change thresholds (1.3 and 0.25, respectively).Green points meet log2 fold change threshold, blue points meet -log10 p-value threshold, and red points meet both log2 fold change and log10 p-value thresholds.Key components of the mitochondrial respiratory chain complex I (MT-ND1, MT-ND4) and complex IV (MT-CO3), and electron transport-coupled proton transport (MT-CO1, MT-CYB) were significantly down-regulated in both RP13 cone and rod photoreceptors.This could reflect reduced mitochondria incidence in RP13 photoreceptors, or a more general mitochondrial dysfunction shown in panel G. F, G) Enriched disease processes and canonical pathways respectively in RP13 cone and rod photoreceptors revealed by the Ingenuity Pathway Analysis (IPA) analysis.The dot colour indicates the z-score assigned by Ingenuity Pathway Analysis to each pathway, where no z-score could be assigned the dot is coloured grey.

Figure S6
. Generation and characterisation of RP13 KiOs.A) Schematic illustration of iPSCs differentiation into KiOs in six-well plates followed by culture in inserts on air-liquid interphase; B) Schematic illustration of kidney nephron with major markers including NPHS1 (podocytes; red), AQP1 (proximal tubules; green), CDH1 (distal tubules and collecting ducts; cyan); C) These markers were used to stain KiO sections.Scale bars are 500 µm for whole organoids and 20 µm for magnified insets indicated by white frames; D) Quantitative immunofluorescence analysis of differentiation of KiOs, indicating variable levels of efficiency.Data shown as mean ± SEM, n = 3; E) Quantitative immunofluorescence analysis of Ki67, a marker of proliferating cells.All lines other than RP13-2 KiOs display clear tubular structures, likely due to increased cell proliferation leading to compromised differentiation efficiency.Data shown as mean ± SEM, n = 5.Statistical significance was assessed using unpaired two-tailed t-test with Welch's correction, * indicates p < 0.05; F) Cell polarity markers in RP13 KiOs.RP13-Cas9 and RP13 KiOs were assessed for apical cell marker (ezrin, green), basal cell surface marker (collagen IV, green), and tight junction marker (ZO-1, green).Asterisks identify the KiO tubule lumen.There were no differences observed in these markers' localisation between control and patient organoids.Scale bars = 10 µm. Figure S8.Cilia analysis in ROs and KiOs.A) Immunofluorescent staining of ROs sections with the ciliary membrane marker ARL13B (green) and PRPF8 (red; scale bar is 20 µm).Insets show localisation of PRPF8 to the base of cilia.Graphs show decreased cilia length in RP13 RO in comparison to isogenic controls, as well as decreased cilia incidence.Data shown as mean ± SEM. n = 4993.Statistical significance was assessed using two-tailed paired Student's t-tests (results are indicated using * = <0.05,** = <0.01,*** = <0.001,**** = <0.0001);B) Immunofluorescent staining of KiOs sections stained with ARL13B (green) and PRPF8 (red; scale bar is 10 µm).Graphs show no differences in cilia length and incidence in RP13 KiO in comparison to isogenic controls.Data shown as mean ± SEM. n = 7845.Statistical significance was assessed using two-tailed paired Student's t-test.

Figure S9
: PRPF8 p.H2309P mutations alters interaction of PRPF8 with snoRNAs and affects 5'SS recognition.A) RP13 mutations affect interaction of PRPF8 with small nucleolar RNAs (snoRNAs).snoRNAs are noncoding RNAs processed from introns in higher mammals that occur in two classes, box C/D and H/ACA snoRNAs.Both classes were significantly affected by PRPF8 mutation as shown in the volcano plot; B) Volcano plot showing how binding of individual snoRNA transcripts are affected by RP13 mutation across all tissues (KiOs = blue, ROs = orange, RPE = green, and iPSCs = red).Statistical significance was assessed using paired t-tests; C) Kurtosis of the histograms presented in figure 7B; D, E) Gene set enrichment analysis of differentially bound weak and strong 5'SSs in KiOs (D) and iPSCs (E).Direction down and up refers to positive and negative fold changes in (RP13/RP13-Cas9), respectively; F) Gene sequence plot showing differential inclusion of nucleotides at weak 5'SSs with upregulated or downregulated binding to PRPF8 in RPE, KiO and iPSCs.showing effect of RP13 mutation on PRPF8 mRNA expression across fibroblasts, iPSCs, KiOs, ROs, and RPE cells.Bar charts show the mean mRNA fold change of PRPF8 in RP13 derived cells relative to the paired RP13-Cas9 controls.n =11 for RP13 and n=16 for RP13-Cas9 samples used for wild type, total and mutant PRPF8 quantification in iPSCs; n=11 for RP13 and n=11 for RP13-Cas9 samples used for quantification of the mutant and total PRPF8 transcript in KiOs, n=21 in RP13 and n=16 for RP13-Cas9 samples used for the quantification of wild type PRPF8 transcript in KiOs; n=11 for RP13 and n=16 for RP13-Cas9 samples used for wild type, total and mutant PRPF8 quantification in ROs; n=3 for RP13 and n=3 for RP13-Cas9 samples used for mutant PRPF8 quantification in RPE cells, n=11 for RP13 and n=14 for RP13-Cas9 samples used for total PRPF8 quantification in RPE cells, n=10 for RP13 and n=14 for RP13-Cas9 samples used for wild type PRPF8 quantification in RPE cells.Statistical significance was assessed using a paired t-test (* = <0.05,** = <0.01,*** = <0.001,**** = <0.0001).B) Western blot results showing effect of RP13 mutation on PRPF8 protein expression across fibroblasts, iPSCs, RPE, ROs, and KiOs.Bar charts show the mean (n = 3) expression of PRPF8 after normalisation to vimentin (VIM) expression.Statistical significance was assessed using 1-way ANOVA, no significant differences were observed.

Supplementary data 10: Differences in protein expression between RP13 and RP13-Cas9
derived ROs, RPE, and KiOs, as determined using TMT-labelling and mass spectrometry.Proteins (represented by 2 or more unique peptides) are listed with differential expression (DE) between RP13 and RP13-Cas9 control proteomes of ROs (4535 unique proteins, DE=203), RPE (4574 unique proteins, DE=56) and KiOs (4294 unique proteins, DE=4).GO and KEGG enrichment analyses of these protein lists are presented in separate sheets.Two additional sheets compare RO and RPE protein expression changes with changes in transcript expression.Two-sided t-test was used for statistical analysis.

Derivation and characterisation of RP13-iPSCs
We ascertained three related RP type 13 patients with a PRPF8 c.6926A>C (p.H2309P) heterozygous missense mutation and one patient with a de novo PRPF8 c.6926A>C heterozygous mutation 1 (Supplementary data 1).All patients were characterised by night blindness in the first decade of life.Dermal skin fibroblasts were reprogrammed to iPSCs using a non-integrative RNAbased Sendai virus system (Figure S1A), as previously described 2 .Several clones from each RP13 participant were tested for expression of pluripotent markers (Figure S1B), clearance of transgenes (Figure S1C) and genome stability (Figure S1D).Apart from clone RP13-1A1, which displayed a decreased copy number in the Chr 12p region, all the other clones displayed the expected copy number in the loci that are most susceptible to changes in pluripotent stem cells.Based on these data, one iPSC clone from each participant was selected for further studies and referred to as RP13-1A, 1B, 1C, and 2 as shown in Supplementary data 1.All iPSCs were maintained in Matrigelcoated plates with mTeSR™ Plus media and passaged using Versene (EDTA 0.02%) in 1:3 -1: 6 ratio.All cultures were maintained at 37°C, in a humidified environment, with 5% CO2.Cells were cryopreserved with freezing media containing 90% foetal bovine serum (FBS), 10% dimethyl sulfoxide and 10 µM Y26732.

Sendai Virus Clearance assay
Total RNA was extracted from 5×10 6 iPSCs using TRIzol® reagent following the manufacturer's instructions.Reverse transcription was carried out using 1 µg of total RNA and the SuperScript cDNA Synthesis Kit following the manufacturer's instructions.Polymerase chain reaction (PCR) was carried out using the cDNA from the reverse transcription reaction with GoTaq DNA polymerase.The PCR products were analysed using 2% agarose gel electrophoresis.All primer details are shown in Table S1.

Detection of pluripotency markers by flow cytometry
iPSCs were dissociated by incubation with Accutase for 5 minutes at 37℃.A total of 1x10 6 single cells were resuspended in PBS supplemented with 5% fetal calf serum (FCS) and stained with anti-TRA-1-60 antibody (directly conjugated to FITC, 1:50) and anti-NANOG antibody (directly conjugated to AlexFluor647 conjugate, 1:50) for two hours at 2-8°C followed by PBS washing and subjected to flow cytometry analysis using FACSCanto flow cytometer (BD Biosciences).At least 10,000 cells were acquired, and the data was analysed using the FACSDiva software.
PluriTest analysis 100 ng total RNA extracted from each RP13 and isogenic control iPSCs was used to prepare the GeneChip® for the PluriTest™ analysis to generate a Pluripotency and Novelty Score.The Pluripotency Score is based on many samples (pluripotent, somatic, and tissues) in the stem cell model matrix which consists of an extensive reference set of >450 cell/tissue types, including 223 hESC lines, 41 iPSCs, somatic cells, and tissues.Samples with positive Pluripotency Scores are more similar to pluripotent samples in the model matrix than to all other classes of samples.The Novelty Score is based on well-characterised iPSCs in the stem cell model matrix.

Genome stability assays
Genome stability was assessed using the hPSC Genetic Analysis kit or the Karyostat assay (Thermofisher).hPSC Genetic Analysis Kit: Genomic DNA from the patient iPSCs was harvested using genomic DNA extraction kit.Genomic DNA was mixed with 2x qPCR master mix and ROX reference dye for qPCR analysis.Nine pairs of primers were applied for the examination of Chromosome regions 1q, 4p, 8q, 10p, 12p, 12q, 18q, 20q and Xp.qPCR ∆∆Ct values were normalised to normal control iPSCs.A copy number < 1.8 or > 2.2 with a p-value <0.05 may indicate the presence of a cytogenetic abnormality.Karyostat Assay: To verify the genome stability of CRISPR/Cas9 isogenic controls, 250ng total genomic DNA extracted from each cell line was used in GeneChip® for KaryoStat™ assays for assessment of potential copy number variants or single nucleotide polymorphisms across the genome.The KaryoStat™ assay allows digital visualisation of chromosome aberrations with a resolution similar to G-band karyotyping.The size of structural aberration that can be detected is >2Mb for chromosomal gains and >1Mb for chromosomal losses.The KaryoStat™ array is optimised for balanced whole-genome coverage with a low-resolution DNA copy number analysis.The assay covers all 36,000 RefSeq genes, including 14,000 OMIM® targets, allowing the detection of aneuploidies, submicroscopic aberrations, and mosaic events.

Correction of PRPF8 p.H2309P mutation in RP13-iPSCs using CRISPR/Cas9
To fully understand the impact of PRPF8 mutation in RPE and retinal cells, CRISPR/Cas9 genome editing was used to correct the PRPF8 c.6926A>C genetic mutation in all patient iPSCs as previously described 2 .For in situ gene correction, a guide RNA was designed to include the PRPF8 c. 6926A>C mutation in the 3' region as well as an asymmetric ssODN template with wild-type PRPF8 sequence with the PAM sequence silenced to prevent repeated cleavage by Cas9 (Figure S2A). 100 iPSC clones from each patient were selected and tested by restriction site analysis using ApaL1, which cleaves the wild-type sequence "GTGCAC".Approximately 20% of the clones showed complete digestion by ApaL1 (Figure S2B): these were further analysed by Sanger sequencing, which demonstrated the correction of the heterozygous mutation "A/C" to homozygous wild type "A" (Figure S2C).To exclude possible off-targeting effects, we tested five candidate genomic regions containing sequences partially matched to the gRNA (Table S1).All the candidate regions showed DNA sequences that match the wild type without insertion or deletion, indicating that no off-targeting occurred during the CRISPR/Cas9 genome editing procedure (Figure S2D).KaryoStat™ analysis, which assesses copy number variants and single nucleotide polymorphisms across the genome (Figure S2E) revealed the lack of genomic instabilities in the corrected clones.The CRISPR/Cas9 RP13-iPSC clones displayed high pluripotency scores in the PluriTest™ analysis (Figure S2F), confirming their pluripotent phenotype.From herein, unedited iPSCs that harbour the pathogenic c.6926A>C mutation in PRPF8 are collectively referred to as "RP13".Similarly, the edited isogenic controls (c.6926C>A) are referred to as "RP13-Cas9".

Differentiation of iPSCs to RPE Cells
Differentiation was performed as previously described 3 .In brief, RP13 and RP13-Cas9 iPSCs were grown on 6 well plates coated with growth factor-reduced Matrigel™, as recommended by the manufacturer's instructions.Cells were incubated at 37⁰C, 5% CO2 and fed every second day with 2 ml mTeSR™ Plus per well.Once iPSCs achieved 80-95% confluency, mTeSR™ Plus media was replaced with 3 ml of differentiation medium (DMEM/F-12, 50 μM β-mercaptoethanol, 1X MEM NEAA, 1X penicillin streptomycin, 20% knockout serum replacement and 10 mM nicotinamide).Differentiation media was partially changed every second day.On days 8 and day 14 media composition was adjusted, by omitting nicotinamide and adding 100 ng/ml Activin A or 3 µM CHIR99021, respectively (Figure 1A).RPE patches formed during this time and were manually excised using a scalpel and transferred to a Falcon tube containing differentiation media.Next, media was aspirated, and patches were washed quickly with PBS.
Then patches were dissociated by incubation at 37°C with TryplE Select (10X) for approximately 20 minutes followed by passage through a 40 µm cell strainer.TryplE Select was neutralised with RPE media and cells were centrifuged for 5 minutes at 300 x g.Media was aspirated and replaced with fresh RPE maintenance media (DMEM/F-12, 50 µM β-mercaptoethanol, 1X MEM NEAA, 1X penicillin streptomycin, and 4% knockout serum replacement).iPSC-RPE were counted using a haemocytometer and plated at density of 1.5x10 5 /cm 2 on 6 well plates coated with growth factorreduced Matrigel™ in 3 ml RPE maintenance media.Media was partially replaced every 2-3 days until cells were pigmented and had a typical "cobblestone" epithelial cell morphology.Cells were then dissociated using TryplE Select (10X) and plated onto 24-well and 12-well PET 0.4 µm pore Transwell ThinCerts™.

Immunofluorescent Staining of iPSC-RPE cells
iPSC-RPE cells grown on 24-well ThinCerts™ were washed with PBS and incubated with 4% paraformaldehyde (PFA) for 20 minutes at room temperature.PFA was then removed, cells were rinsed twice with PBS and stored in PBS at 4°C until required.The transwell culture surface was isolated and placed on Superfrost+ microscope slide.Pigmentation of RPE cells was removed using Melanin Bleach Kit, as recommended by the manufacturer.RPE cells were blocked in PBS supplemented with 10% normal donkey serum and 0.3% Triton-X100, for 1 hour at room temperature.For ezrin immunostaining, RPE cells were not bleached, and Triton X-100 was substituted for a 5-minute incubation at -20°C with methanol, prior to blocking.The RPE cells were incubated with primary antibodies overnight at 4°C.Following three washing with PBS, RPE cells were incubated with secondary antibodies diluted in antibody dilution (PBS and 1% normal donkey serum) with Hoechst for 1 hour at room temperature.Then, iPSC-RPE cells were washed 3X for 5 minutes with PBS before mounting with Vectashield and sealed with a #1 coverslip.RPE cells were imaged using an AxioImager upright microscope with Apotome structured illumination fluorescence using 20x, 40x and 63x oil objectives (Zeiss, Germany).Three to five images per patient were processed using ZEN 2.5 Lite software (Zeiss).For cilia studies, RPE cells were imaged using an 100x objective on an A1R confocal microscope (Nikon).Images were analysed using Fiji (ImageLab) to assess co-localisation of ciliary proteins, and Huygens software (Scientific Volume Imaging) to determine cilia length.Data were analyzed and processed in Prism 7 (GraphPad).

Measurement of trans-epithelial resistance (TEER)
Trans-epithelial resistance (TEER) was measured in Transwells containing RPE cells by inserting the tip of the short electrode in the inner side of the Transwell insert, and the tip of the longer electrode in the outer side of the Transwell insert.Also, the resistance of a blank TW containing PBS was measured.Three measurements were obtained from each Transwell insert.After that, the readings from the blank Transwell were subtracted from the readings obtained from Transwells containing RPE cells.Based on the formula Unit area resistance = Resistance (Ω) x Effective Membrane Area (cm2), the results were multiplied by 0.3, which is the membrane area of a Transwell, and final values were given in ohms (Ω).TEER measurements were performed one day after feeding the RPE cells to avoid variability in the TEER readings.

Phagocytosis assay
Bovine rod photoreceptor outer segments (POSs) were centrifuged at 2600 × g for 4 minutes, and the pellet was resuspended with 0.4 mg/ml FITC for 1 hour at room temperature followed by agitation in the dark.Following centrifugation at 2600 × g for 4 minutes and washing of the POSs three times with PBS, POSs were resuspended in AdRPMI 1640 media supplemented with B-27 Supplement and FBS.The staining of POSs was confirmed under a Bioscience Axiovert microscope.RPE cells were treated with unlabelled and FITC-labelled POSs (20 POSs per cell) and incubated for 4 hours at 37°C.For control, RPE cells were treated with FITC-labelled POSs and were incubated at 4°C for 4 hours.Both control and RP13-RPE cells were washed with PBS and were detached from the wells using 200μl of TryplE Select (10X) for 15 minutes.Then, control and RP13-RPE cells were resuspended in flow buffer (PBS with 2% FBS) and were transferred into Eppendorf tubes followed by centrifugation at 300 × g.To distinguish cells from debris and unbound POSs, RPE cells were incubated with 5 mM DRAQ5 for 5 minutes.To quench fluorescence from unbound POSs, cells were incubated with 0.2% Trypan Blue solution for 10 minutes.RPE cells were washed 3 times with PBS, and cell pellets were resuspended in flow buffer.Samples were run on a LSRII flow cytometer, and 10,000 events were collected per sample.Results were analysed using FacsDiva software.

RPE cytokine secretion studies
Medium from basal and apical chambers of transwell inserts were collected from RPE cells of healthy controls and patients.The levels of PEDF and VEGF secretion were measured by using human PEDF-ELISA Kit (Cusabio, CSB-E08818h) and human VEGF-ELISA Kit (Life Technologies) according to the manufacturer's instructions.
Immunofluorescent Staining of iPSCs-ROs 10 µm thickness sections of ROs were collected on glass cover slips and were separated using an ImmEdge pen.Sections were air-dried for 20 minutes at room temperature and washed with PBS to remove OCT 91 from the tissue.ROs sections were incubated with a blocking solution (5% normal goat or donkey serum, 0.3% Triton-X in PBS) for 1 hour at room temperature, followed by overnight incubation with primary polyclonal non-conjugated antibodies shown in the Key Resources Table at 4°C.Primary antibodies were diluted in antibody diluent solution containing PBS, 1% bovine serum albumin (BSA) and 0.3% Triton-X.Following overnight incubation, retinal sections were washed three times with PBS for 15 minutes each and incubated with secondary antibodies conjugated to fluorophores and diluted in antibody diluent for 1 hour at room temperature.RO sections were then washed three times with PBS for 10 minutes each and covered by Hoechst nuclear stain diluted in Vectashield at 1:2000, followed by mounting of the slides by a coverslip.

KiOs differentiation from iPSCs
Kidney cell and organoid culture was based on established protocols 4 .RP13-and RP13-Cas9-iPSCs were cultured in mTeSR™ Plus.To start differentiation into kidney cells, iPSCs were dissociated into single cells with TryplE.400,000 cells were plated in Matrigel-coated 6-well plate with 2 ml mTeSR™ Plus containing 10 μM ROCK inhibitor (Y-27632) and incubated overnight (day -1).The next day, cells were at 50% confluency and media was changed to APEL2 containing 5% protein-free hybridoma medium (PFHM-II) (complete medium; henceforth called APEL2+) and 8 μM CHIR99021 (day 0).The same media change was done on day 2. On day 4 of differentiation, media was changed to APEL2+ containing 200 ng/ml FGF9 and 1 μg/ml heparin.Media was changed every other day.On day 7 of differentiation, kidney cells were trypsinised, resuspended into a single cell suspension and counted.500,000 cells were transferred to an Eppendorf tube and spun down at 800 x g for 3 minutes at room temperature three times, with gentle tube agitation after each spin.3D KiO cultures were grown on an air-liquid interphase, on cell culture inserts called Thincerts (Greiner Bio-One Ltd).Kidney cell pellets were transferred into the inner part of the Thincert, and KiOs were allowed to mature.APEL2+ containing 5 μM CHIR99021 was added to the outer part of the Thincert and incubated for 1 hour at 37°C, following media change to APEL2+ containing 1x antibiotic/antimycotic (complete media; henceforth called APEL2++), 200 ng/ml FGF9 and 1 μg/ml heparin.Media containing growth factors was changed every other day throughout the differentiation.Growth factors were withdrawn on day 12 and culture with APEL2++ media was carried on until day 25.

Cyst induction
A 24-well plate was coated with Lipidure by placing 500 μl of 0.5% w/v Lipidure solution in each well and allowing complete evaporation at room temperature in sterile conditions.On day 22, KiOs were moved from Thincerts into a non-adherent plate with APEL2++ media containing 30 μM forskolin, 12.5 μM blebbistatin or DMSO.Organoids were grown in suspension with drugs for 72 hours.

KiOs processing
On day 25 organoids were washed with PBS and snap-frozen in liquid nitrogen for biochemical and proteomic assays.Organoids for RNA sequencing were suspended in 1 ml Trizol and snap-frozen.Organoids for immunofluorescent staining were fixed with 4% PFA for a minimum of 4 hours, followed by three PBS washes.They were permeabilized and blocked using 2% BSA in PBS containing 0.3% Triton X-100 overnight at 4°C.After three PBS washes, organoids were incubated in primary antibodies diluted in 1% BSA overnight at 4°C.Following three PBS washes, organoids were incubated in secondary antibodies diluted in 1% BSA overnight at 4°C.On the final day, organoids were washed three times with PBS and imaged with a confocal A1R microscope (Nikon) at x4 magnification.KiOs for OCT embedding were fixed with 4% PFA, washed with PBS and soaked in 30% (w/v) sterile sucrose overnight at 4°C on a roller.Following a PBS wash, organoids were placed in moulds, submerged in OCT and frozen.Frozen OCT blocks were cryosectioned at 10 μm thickness on a Leica CM1950 cryostat.Sectioned organoids were stored at -80°C until use.Further processing is described below.Images were processed in Fiji (ImageJ).Image processing included the use of the following plugins: "Colocalisation Threshold," "CiliaQ" and "Mitochondria Analyser."

Transmission Electron Microscopy (TEM)
Trans-wells of RPE cells, ROs and KiOs were washed with PBS and fixed overnight with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C.The samples were post-fixed in 1% osmium tetroxide, dehydrated in an acetone gradient, and embedded in epoxy resin.Ultrathin sections of 70 nm thickness were picked up on copper grids, stained with uranyl acetate and lead citrate and imaged using a Philips CM100 Hitachi HT7800 transmission electron microscope with high-resolution digital image capture.For iPSC-RPE, ten cuboidal cells were images per sample at a magnification of 5000x.Sample identifications were created using a random number generator, images were then sent to researchers who were blinded to PRPF8 mutation status.Melanosomes and vacuoles were counted manually by first importing the images into Microsoft PowerPoint.All other measurements were taken using Microscopy Image Browser (MIB) 5 .

Cilia Length and Frequency Measurements in RPE Cells, ROs, and KiOs
Samples were blocked in 1% non-fat milk in PBS for 30 minutes then stained with ARL13B primary antibody.Images were obtained using a Nikon A1R confocal microscope with ×100 oil objective lens controlled by NIS Elements AR 4.20.01(Nikon) software.Optical sections were generated through structured processing using NIS Elements AR 4.20.01(Nikon) software.Images were deconvoluted using the deconvolution wizard and batch processor in Huygens software (Scientific Volume Imaging).ARL13B objects were segmented automatically in Huygens and the object analyser was used to calculate ARL13B object length.A minimum of 500 cilia from at least three non-overlapping fields of view were analysed per sample.Nuclei were counted manually, and data was then used to calculate ciliary incidence.

Serial block face scanning electron microscopy (SBF-SEM)
RPE cells were fixed overnight in 2% glutaraldehyde in 0.1M sodium cacodylate buffer.Once fixed, the samples were processed using an adapted heavy metal staining protocol 6 .Briefly, samples were incubated in a series of heavy metal solutions -3% potassium ferrocyanide in 2% osmium tetroxide, 10% thiocarbohydrazide, 2% osmium tetroxide again, 1% uranyl acetate overnight, and finally lead aspartate solution.Between each step the samples were rinsed thoroughly in several changes of deionised water.Samples were dehydrated through an acetone gradient and then impregnated with increasing concentrations of Taab 812 hard resin, with several changes of 100% resin.The samples were flat embedded in 100% resin and left to polymerise at 60°C for a minimum of 36 hours.The resin blocks were trimmed to approximately 0.75 mm by 0.5 mm and glued onto an aluminium pin.To reduce sample charging within the SEM, the block was painted with silver glue and sputtercoated with a 5 nm layer of gold.The pin was placed into a Zeiss Sigma SEM incorporating the Gatan 3view system, which allows sectioning of the block in situ and the collection of a series of images in the z-direction.Multiple regions of interest were imaged at x2000 magnification, 3000 x 1500 pixels scan, which gave a pixel resolution of approximately 15 nm.Section thickness was 100 nm in the z-direction.The resulting z-stacks were post processed in MIB using batch processing and cilia were identified and segmented manually using MIB.The segmentations were imported into Amira (FEI) for construction of the 3D models.

Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted using the ReliaPrep RNA Cell Miniprep System according to the manufacturer's protocol.One μg of RNA was reverse transcribed into cDNA with random primer by reverse transcriptase (RT).Relative gene expression was determined by quantitative real-time RT-PCR, using 20 ng cDNA (relative to RNA amount) for each sample with the SYBR Green Master Mix in a 10μl reaction.Ct values were measured using QuantStudio™ 7 Flex Real-Time PCR System (ThermoFisher).Primers against GAPDH were used as the endogenous control to normalize the amounts of RNA in each sample.Primer sequences are shown in Table S1.

RNA extraction and RNA-Seq
Trizol suspensions of iPSCs, RPEs, KiOs and ROs were homogenized using 26-gauge syringe needles followed by standard Trizol RNA extraction.Extracted RNA was further purified using DNAse TURBO, and RNA purity and concentration was evaluated on TapeStation (Agilent Technologies).RNA libraries were prepared using TruSeq Stranded mRNA libraries and a total of 105 samples was run on four lanes of NextSeq 2000 at 200 cycles 100bp paired end (each tissue type was run per lane).

Single cell RNA-Seq
ROs samples at day 210 of differentiation were dissociated to single cells using a neurosphere dissociation kit (Miltenyi Biotech).10,000 cells from each sample were captured, and sequencing libraries generated using the Chromium Single Cell 3' Library & Gel Bead Kit (version 3.1.10x Genomics) for scRNA-Seq.Single cell RNA-Seq libraries were sequenced to 50,000 reads per cell on an Illumina NovaSeq 6000.The 8 samples were aligned to human reference genome (GRCh38) using CellRanger Version 3.0.1.Dead cells and debris were removed from downstream analysis by excluding cells with a minimum of 2000 counts per cell, a minimum of 500 genes per cell and a maximum of 20% mitochondrial reads per cell.Doublets were removed using DoubletFinder.A standard Seurat (version 4.3) clustering workflow was followed to identify clusters in each cell.Default parameters were used for the NormalizeData, FindVariableFeatures, ScaleData, RunPCA, and FindNeighbors.Each sample was clustered at a resolution of 1 with the FindClusters function and a UMAP plot using the first 20 principal components was used to visualise the data.To create a combined dataset each sample was downsampled to 5500 to ensure that clustering was not driven by cell number.There were small technical differences between the number of counts and number of genes and percentage mitochondria between the three samples.These factors were regressed out using the ScaleData function and Harmony batch correction applied.The data was reclustered using the above method.The first 10 principal components were used for FindNeighbors and RunUMAP functions.We tested a range of clustering resolutions from 0.2 -2.2.Differentially expressed markers for each cluster were obtained using the FindAllMarkers function for each resolution.Cell types were annotated using known marker genes.

Bulk RNA-Seq Data analyses
Quality control and alignment against the reference genome: RNA paired-end sequencing quality control was assessed using FastQC 7 and multiQC 8 giving on average 76 million mapped reads (Table S3).Both adapters and low-quality bases (QV < 20) were trimmed from read extremities using Trimmomatic 9 .All libraries were mapped against the Human GRCh38 release-105 reference genome (retrieved from http://ftp.ensembl.org/pub/release-105/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz) through the STAR aligner 10 with default parameters.STAR-generated sorted BAM output files were used for assigning read counts to gene features with featureCounts 11 with the following parameters: -p -B -C -M -O --fraction -s2 and using Homo_sapiens.GRCh38.105.gtfannotation file downloaded from the same Ensembl ftp address as above.

Differential gene expression:
Read counts tables generated by featureCounts and Libinorm 12 were used as input for differential expression (DE) analyses using DeSeq2 13 .DE pairwise comparisons were performed either for each cell type per participant, or for each cell type per pooled participant samples.In all analyses, the fold-change ratio was expressed as mutant RP13: isogenic control RP13-Cas9.The same read count table analyzed for multi-dimensional scaling (MDS) using the plotMDS function in the EdgeR package 14 .EnhancedVolcano (https://bioconductor.org/packages/release/bioc/html/EnhancedVolcano.html) was employed for an overall DE visualization through volcano plots.Tools were run under the R environment, version 4.1.0.Evaluation of Differential Splicing: STAR-generated sorted BAM files were used as input for rMATs turbo v4.1.2 15for differential splicing (DS) analyses under the following parameters: --libType fr-firststrand --readLength 88 -variable-read-length -novelSS.The pairwise comparisons of sample groups for DS analyses followed the same rationale as for DE described above.We wrote a PERL script (https://github.com/eltonjrv/bioinfo.scripts/blob/master/filter_rMATs.pl) to filter rMATs results according to our set of pre-established thresholds: FDR < 0.05, Inclusion Level Difference (IncLevelDifference) > 0.05, and both inclusion and skipping junction counts average (IJC_SJC_avg) > 5. DEXSeq 16 was also used with default parameters for differential exon usage (DEU) score calculations, following the same pairwise comparisons as for both DE and DS analyses.Uniquely mapped, junction-spanning reads were used by MAJIQ to construct splice graphs for transcripts from a custom Ensembl transcriptome annotation, and to quantify PSI and ΔPSI for all local splicing variations (LSVs).The captured LSVs include classical alternative splicing events (e.g.cassette exons, alternative 5'SSs, etc.) as well as more complex variations 17 .In addition, we employed SUPPA 18 and MAJIQ 19 to determine both PSI values (within conditions) and ΔPSI values (between conditions).Regional Coverage was counted using Sailfish quant 12 followed by the tools described above.The differences in variation of were analysed using an f-test for each event, and a t-test was used to compare the log10 normalized value of the whole dataset per tissue.f-tests and t-tests were conducted using custom code written in SciPy 20 .

Gene enrichment functional analysis
Gene lists of differentially expressed genes at DE or DS levels were enriched using GSEApy 21 , using the Genetic Ontology (GO) 22 and Reactome 23 libraries.GO-based gene enrichment analyses were performed by clusterProfiler Bioconductor package 24 under R v4.1.0,set to an adjusted pvalue < 0.05.We limited the background gene set to only include those genes that are expressed in the relevant RNA-seq dataset for each cell type to improve the accuracy of identifying enriched pathways altered between cell types and conditions.Alphafold2 analysis of predicted protein structures PRPF8 protein sequence (XP_024306305.1)and mutant versions were folded using ColabFold 25 using an Alphafold2 backend 26 , running in single sequence mode (alphafold2_ptm model) and five recycling steps.We generated 25 replicates with differing seeds to average out small folding variations.All models were rotated and translated to fit an arbitrarily chosen model (wildtype with seed 001) before averaging them.Functional regions were obtained from previous mutational studies 27 .

TMT labelling for mass spectrometry
Total cell lysates were prepared from RP13 and RP13-Cas9 ROs, KiOs, IPSCs or RPE cells following the instructions of the TMT10plex™ Isobaric Mass Tagging Kit (Thermo Scientific).After pipetting cells in the lysis buffer and heating at 95°C for 5 minutes, lysates were diluted to 120 μl and sonicated.Protein concentrations were determined using the Pierce BCA protein assay kit (Thermo Scientific).To this end, 100 μg proteins from each sample were reduced by the addition of tris(2-carboxyethyl)phosphine, alkylated with iodoacetamide followed by acetone precipitation.Protein pellets were resuspended in 50mM triethyl ammonium bicarbonate (TEAB) buffer and were digested with trypsin overnight at 37 °C.Peptides from each sample were then labelled with a specific TMT reagent for 1 hour at room temperature.Reactions were quenched by 5% hydroxylamine for 15 minutes.Fifty micrograms of TMT-labelled peptides from RP13 and control samples were combined and cleaned up using C18 spin columns (Harvard Apparatus) and subjected to pre-fractionation using the high-pH reversed-phase peptide fractionation kit following the manufacturer's instruction (Thermo Scientific).Collected fractions were dried and resuspended in 50 μl of 0.1% formic acid for mass spectrometry analysis.

LC-MS/MS analysis
Peptides in each fraction were analysed in two injection replicates using either an Orbitrap Fusion or a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific), both coupled with an UltiMate 3000 RSLCnano HPLC system (Thermo Fisher Scientific), as previously described 2 .
Data processing MS/MS spectra were searched against a SwissProt human database containing 20,341 reviewed protein entries using the MASCOT algorithm (Matrix Science) in Proteome Discoverer 2.2 (PD, Thermo Fisher Scientific), and were processed as previously described 2 .At least two quantifiable unique peptides in each replicate were required for protein quantification.Protein ratios were logtransformed and then median normalised using the Perseus software version 2.0.6.0 28 .The reported RP13: RP13-Cas9 (mutant : control) ratios are the average of two replicates.To identify the differentially expressed proteins, those proteins with a log2-fold change (LFC) less than −0.5 or greater than +0.5 and a p-value <0.05 were defined as differentially expressed (DE).Gene Ontology (GO) enrichment analyses were carried out by ShinyGo (FDR cut-off 0.05).

Western blot
We have used two methods of Western blot, for PRPF8 protein levels across different tissues we used LI-COR reagents, for the remaining protein levels analysis we used ECL method.Cells were harvested and washed with cold PBS, re-suspended in lysis buffer (25mM Tris-Cl pH 7.5, 120 mM NaCl, 1 mM EDTA pH 8.0, 0.5% Triton X100) supplemented with protease inhibitors (Roche 11697498001) and lysed by ultra-sonication (twice, for 6 seconds) (Bradson Sonifier150) to obtain whole cell lysate.The protein concentration was determined using the Bradford Dye Reagent (Bio-Rad 500-0205).Fifty μg of whole lysate was applied to SDS-PAGE and transferred to Hybond PVDF membrane (GE Health 15259894), followed by standard western blot procedure.The bound primary antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibody and the ECL detection system (GE Health GERPN2232).
Proteins were extracted from cells and organoids using NP40 lysis buffer containing protease inhibitors.Protein extracts were sonicated five times for 5 seconds at 5 mA, with samples kept on ice at all times.Sonicated protein extracts were incubated on a rotator at 4 o C for 30 minutes, followed by centrifugation at 15,000 x g for 15 minutes at 4 o C. Protein concentration was determined using the DC™ Protein Assay Kit II.Ten μg of protein extract was loaded on 3-8% Tris-Acetate gel and run for 1 hour and 20 minutes at 150V.Proteins were then transferred on PVDF membrane overnight at 25V at 4 o C. Membrane was blocked in Intercept Blocking Buffer for 1 hour at room temperature and incubated with primary antibodies overnight on a roller at 4 o C.After four 1xPBS washes membrane was incubated with secondary antibodies for 1 hour at room temperature in the dark and again washed four times with 1xPBS.After one dH2O wash, the membrane was dried by sandwiching between two pieces of blotting paper.Dried membranes were then imaged on LI-COR, followed by band intensity calculations.

Analysis of snRNPs Levels by Glycerol Gradient Fractionation and Northern blotting
Analysis of snRNPs levels were performed according to our previous protocol 29 with minor modifications.Briefly, RPE cells were washed with PBS, harvested by centrifugation and pellets were resuspended in 20 mM HEPES-KOH, pH 7.9, 100 mM KCl, 10% (v/v) glycerol, 0.2 mM EDTA, 1 mM DTT. Whole-cell extracts were prepared using syringes with 18-gauge needles followed by sonication (three times bursts at 30% for 5 seconds on ice, and 30 seconds pauses in between bursts).Sonicated lysates were cleared by centrifugation at 15,000 x g for 20 minutes at 4°C, and were aliquoted, flash frozen and stored at -80°C.Whole-cell extracts were sedimented on linear 4 ml 10-30% (v/v) glycerol gradients in the G150 buffer (20 mM HEPES pH 7.9, 150 Mm NaCl, 1.5 mM MgCl2 and 0.5 mM DTT).After ultracentrifugation in a Sorvall TH-660 rotor for 14 hours at 29,000 rpm (114136.7 x g), the gradients were fractionated into 24 fractions.To analyse the relative levels of snRNPs, proteins in the odd-numbered gradient fractions were digested with proteinase K in 20 mM HEPES-KOH pH 7.9, 150 mM NaCl, 10 mM EDTA and 1% (w/v) SDS for 45 minutes at 42°C.The total RNAs were extracted with phenol/chloroform/isoamylalcohol and were precipitated with sodium acetate and ethanol at -20°C for 2 hours.The isolated RNAs were separated by denaturing 8% urea PAGE followed by Northern blotting and hybridisation with 5'-end biotinylated probes against U4, U6 and U5 snRNAs 30 .The biotin-labelled probes were detected using the chemiluminescent nucleic acid detection module kit (Thermo Scientific) according to the manufacturer's instructions.To analyse the distribution of selected splicing proteins across the gradients, proteins were acetone precipitated from even-numbered gradient fractions and separated on NuPAGE 4−12% Bis-Tris gels (Invitrogen) followed by Western blotting on a nitrocellulose membrane (GE Healthcare).Immunostaining for PRPF8, Brr2 (SNRNP200), Snu114 and PRPF31 was performed using specific antibodies and the Amersham ECL detection kit (GE Healthcare).All antibody details are shown in the Key Resource Table .Fluorescence in situ hybridisation Fluorescence in situ hybridisation (FISH) was performed using U2, U4, U5 or U6 snRNA probes labelled with AlexaFluor647 or an oligo dT(45) probe labelled with ATTO488 at the 5'-end.The sequences of snRNA probes and the RNA-FISH method were as previously described 29,30 .After FISH, for snRNA probing, cells were immunostained for coilin.For poly-A + RNA FISH, cells were immunostained for SC35 and prepared for confocal microscopy as described in the methods sections named immunofluorescent staining of RPE cells and ROs.Quantification of mean intensities was performed with the ImageJ/Fiji software from three independent measurements.

Co-localisation of SC35 and PRPF8 -Image analysis
Co-localization analysis of SC35 and PRPF8 immunofluorescence images was performed using ImageJ/Fiji software.Each coloured image was separated into individual channels; red (for PRPF8), green (for SC35), and blue (Hoechst).For each dye, the same sized region of interest (ROI) was selected for further analysis.The degree of co-localization was calculated as mean of the correlated pixel intensities of the red and green channels, using the embedded co-localization analysis plug-in at default settings.iCLIP-seq library preparation PRPF8-RNA interactions were stabilized by UV crosslinking (254 nm, 400 mJ/cm 2 ) of cells, followed by lysis in 1 mL of iCLIP lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630 (Sigma I8896), 0.1% SDS , 0.5% sodium deoxycholate), limited digestion with RNase I (0.8 units/ml), immunoprecipitation of PRPF8-RNA complexes with anti-PRPF8 antibody at 10 μg per mL of lysate and 150 μl magnetic protein A Dynabeads, followed by stringent washes.After dephosphorylation with FastAP alkaline phosphatase and T4 Polynucleotide Kinase, a barcoded pre-adenylated adaptor (0.1 μM) was ligated to the 3′ end of precipitated RNA using T4 RNA ligase I (3 units/μl).Ligation was performed on-bead in 20% PEG8000 to improve ligation efficiency.Unincorporated adaptor was removed by treatment with 5' deadenylase (1.25 U/μl), RecJf endonuclease (0.75 U/μl) and stringent washes.PRPF8-RNA complexes were eluted from Dynabeads using preheated 1x NuPAGE loading buffer at 70℃ and loaded onto a 4-12% NuPAGE Bis-Tris gel running for 65 minutes at 180V.After transfer to a nitrocellulose membrane (30V, 2 hours), a region 75 kDa (~220 nt of RNA) above PRPF8 (274 kDa) was excised from the membrane and treated with proteinase K to isolate RNA.The RNA was reverse transcribed by Superscript IV reverse transcriptase using irCLIP_ddRT primers (1pmol/μl) and circularized using CircLigase II.The cDNA was PCR amplified for 21 cycles with Platinum™ II Hot-Start PCR Master Mix using P5Solexa/P3Solexa primers.The PCR product was size selected using agarose gel electrophoresis, and the 150-400bp region was excised for gel extraction followed by purification with AMpure XP.This was then sequenced by using the Illumina sequencing platform (See Table S1 for oligonucleotide sequences and Key Resource Table for catalogue numbers of all reagents).iCLIP analysis: FastQC 7 was used for quality control of iCLIP-seq data similar to the raw data from RNA-seq.Reads were mapped using STAR 10 onto GRCh38 and duplicate reads were filtered by UMItools 31 .Events were quantified by assessing the highest peak within annotated regions (GRCh38), rather than RPKM, to identify peak locations without averaging them out.Significant differential events were normalised and quantified using DESeq2 13 .Significant events were annotated by RNA biotype using the RNAcentral database 32 .We assessed splice sites using maxentpy/MaxEntScan 33 to calculate the 3' and 5'SS strength of exons corresponding to each event, based on the gene annotation in GRCh38.

Figure S2 :
Figure S2: PRPF8 mutation correction by CRISPR/Cas9 gene editing.A) Diagram showing the design of asymmetric ssODN aligned to the PRPF8 genomic sequence and guide RNA (gRNA; cyan); B) ApaL1 restriction enzyme analysis showing the digest of 602 bp fragment into 206 and 396 bp band in the successfully corrected clones; C) Representative DNA sequence of RP13-iPSC and isogenic CRISPR/Cas9 control showing the correction of heterozygous mutation to the homozygous wild type nucleotide: N indicates where a nucleotide was not automatically assigned, and R indicates the base could be either C or A; D) Representative DNA sequences of all five tested off-target sites showing no deviations from the wild type sequence; E) Karyostat genome stability assay excluded chromosomal aberrations in CRISPR/Cas9 corrected iPSC lines.The whole genome view displays all somatic and sex chromosomes in one frame with high level copy number.A value of 2 represents a normal copy number state (CN = 2), value of 3 represents chromosomal gain (CN = 3), value of 1 represents a chromosomal loss (CN = 1).The pink, green and yellow colours indicate the raw signal for each individual chromosome probe, while the blue represents the normalized probe signal used to identify copy number and aberrations (if any); F, G) Tableandaccompanying plot of results from PluriTest assay.The red and blue areas of the plot show how the tested samples compare with reference pluripotent and non-pluripotent cell types, respectively.PluriCor and NovCor were determined using an algorithm that integrates gene expression data to authenticate pluripotency status.In brief, PluriCor is a score of pluripotency whereas NovCor increases when non-pluripotent cell types are also present.A non-iPSC sample was used in this experiment to serve as a negative control for non-pluripotency.

Figure S7 :
Figure S7: Analysis of PRPF8 localisation to splicing speckles.A) Co-staining of PRPF8 (red) and splicing speckles (SC35, green) followed by counterstaining by DAPI in RPE and ROs showing localisation of PRPF8 to the speckles of RP13 and equivalent Cas9 controls.The inset shows the magnification of the selected region; B) Confocal microscopy analyses of RNA-FISH labelling for U5 snRNA (red) in Cajal bodies (anti-coilin, green) in control and RP13-RPE cells.Arrows indicate CBs.Scale bar is 10 µm; C) Immunostaining of iPSCs, KiOs, RPE and ROs with SC35 (SRSF2), a marker for nuclear speckles, showing dispersion of nuclear speckles (white arrows) only in RP13photoreceptor and RPE cells.Scale bar is 10 µm.D) Docking of the U6ISL and U6/5'SS to PRPF8 and the location of crosslinked U6 nucleotides (U64-G65) in the structure of Bact spliceosome.The PRPF8 domain organisation is shown at the top and similar colour codes have been used in the PRPF8 3D structure.The U6 ACAGA-box is also labelled.NTD, N-terminal domain; NTDL, NTD linker; HB, helical bundle; RT, reverse transcriptase-like; En, endonuclease-like; RH, RNase H-like domain.E) Profiles of the U5, U4 and Us snRNAs across the gradients shown in Figure 2C.

Figure S10 :
Figure S10: Transcript and protein expression of PRPF8.A) Quantitative PCR (qPCR) datashowing effect of RP13 mutation on PRPF8 mRNA expression across fibroblasts, iPSCs, KiOs, ROs, and RPE cells.Bar charts show the mean mRNA fold change of PRPF8 in RP13 derived cells relative to the paired RP13-Cas9 controls.n =11 for RP13 and n=16 for RP13-Cas9 samples used for wild type, total and mutant PRPF8 quantification in iPSCs; n=11 for RP13 and n=11 for RP13-Cas9 samples used for quantification of the mutant and total PRPF8 transcript in KiOs, n=21 in RP13 and n=16 for RP13-Cas9 samples used for the quantification of wild type PRPF8 transcript in KiOs; n=11 for RP13 and n=16 for RP13-Cas9 samples used for wild type, total and mutant PRPF8 quantification in ROs; n=3 for RP13 and n=3 for RP13-Cas9 samples used for mutant PRPF8 quantification in RPE cells, n=11 for RP13 and n=14 for RP13-Cas9 samples used for total PRPF8 quantification in RPE cells, n=10 for RP13 and n=14 for RP13-Cas9 samples used for wild type PRPF8 quantification in RPE cells.Statistical significance was assessed using a paired t-test (* = <0.05,** = <0.01,*** = <0.001,**** = <0.0001).B) Western blot results showing effect of RP13 mutation on PRPF8 protein expression across fibroblasts, iPSCs, RPE, ROs, and KiOs.Bar charts show the mean (n = 3) expression of PRPF8 after normalisation to vimentin (VIM) expression.Statistical significance was assessed using 1-way ANOVA, no significant differences were observed.